The goal of this proposal is to understand how to efficiently direct differentiation of human embryonic stem cells (HESCs) into different endoderm lineages, such as lung, liver, pancreas and intestine for therapeutic use to replace diseased or damaged tissue. I hypothesize that current ES cell differentiation protocols can be greatly improved by incorporating a critical anterior-posterior (A-P) patterning event that normally occurs during early endoderm organ development. In addition, I also hypothesize that manipulating signals responsible for A-P patterning will allow us to systematically generate the full range of different endoderm lineages including lung, thyroid and intestine. Published studies from our and other labs have demonstrated that controlling the levels of FGF and Wnt signaling prior to formation of a gut tube is critical for patterning the endoderm along the anterior-posterior (A-P) axis and for establishing the organ domains. The endoderm patterning activity of FGF and Wnt signaling pathways is conserved in HESCs. From our preliminary data I hypothesize that the FGF and Wnt pathways synergize to promote human endoderm cell fate in two ways, first by regulating expression of key A-P determinants and second by directing cell migration along the A-P axis. Furthermore I hypothesize that by manipulating the levels of FGF and Wnt signaling I can efficiently direct definitive endoderm (DE) into specific foregut lineages, including pancreas and liver. The proposed experiments will allow, for the first time, interrogation of how FGF and Wnt pathways direct the early stages of foregut specification in humans and to more efficiently direct HESCs into therapeutically important tissues. Moreover, the advantages of a cell culture system will allow me to test mechanistic interactions between FGF and Wnt signaling which are difficult to address in animal models. Specific Aim 1: Determine how FGF and Wnt signaling synergize to pattern human endoderm and direct foregut lineage specification. Specific Aim 2: Investigate the differentiation potential of HESC-derived foregut endoderm in vivo. Specific Aim 3: Determine how FGF4 coordinates human endoderm migration. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to understand how to efficiently direct differentiation of human embryonic stem cells (HESCs) into different endoderm lineages, such as lung, liver, pancreas and intestine for therapeutic use to replace diseased or damaged tissue.